The overall goal of this project is to understand how nucleo-cytoskeletal interactions position nuclei in migrating cells and how the positioning of nuclei affects cell migration. Nuclei are positioned by active mechanisms in virtually all animal cells and occupy specific positions that reflect the activity of the cell type they reside in. Connections between the nucleus and the cytoskeleton are mediated by outer nuclear membrane nesprin proteins, which interact directly with cytoskeletal elements, and SUN proteins in the inner nuclear membrane that anchor the nesprins. In earlier work from our laboratory, we found that the nucleus is actively moved rearward during polarization of fibroblasts for migration into in vitro wounds and that this movement involved the assembly of nesprin2 and SUN2 into novel linear arrays along moving actin filaments that encountered the nucleus. These arrays, which we termed TAN lines for transmembrane actin-associated nuclear lines, tether the nucleus to moving actin filaments and treatments that disrupt TAN lines prevent nuclear movement, centrosome orientation and inhibit migration of cells into in vitro wounds. To understand more about this novel membrane structure and its role in positioning the nucleus for cell migration, we propose to further develop the wounded fibroblast monolayer system to test subcellular mechanisms of nuclear positioning and the role of nuclear positioning in cell migration. In the first aim, we will examine the dynamics of TAN line assembly with live cell imaging experiments, learn more about the structure of TAN lines using electron microscopy and super resolution light microscopy approaches, and study the composition of TAN lines by screening for additional proteins that may contribute to the assembly and/or anchoring of this structure in the nuclear membrane. We will also examine the broader significance of TAN lines for cell migration by testing their role in nuclear positioning in cells migrating in different environments and test the specific hypothesis that TAN lines may be necessary for cells to migrate through small spaces. In the second aim, studies will focus on the question of how nuclear positioning is read out by migrating cells. We will pursue preliminary studies that have identified a separate set of nuclear membrane proteins that appears to act as a nuclear organizer for myosin activation and that when disrupted, results in altered directionality of actin flow in cells and movement of nuclei to eccentric positions. The components of this system will be examined to understand the myosin activation events at the nuclear surface and the manner in which this activation spreads outward to ensure directionality of actin flow in migrating cells. By pursuing these studies, we will learn more about the basic mechanisms by which the nucleus interacts with the cytoskeleton and how this contributes to the polarization of migrating cells. As mutations in genes encoding proteins involved in nuclear positioning pathways cause a number of human diseases, including muscular dystrophies, cardiomyopathies, cerebellar ataxia and lissencephaly, these studies will also provide new avenues for understanding human disease and developing strategies for their treatment.